1. Field of the Invention
Proteins are biologically synthesized macromolecules having various roles in living systems. Enzymes are particular varieties of biologically-active proteins which catalyze specific reactions. Presently, enzyme technology is used in many areas in industry and research, such as, for example, medical research, food processing and preservation, the production of fermented beverages, the production of pharmaceuticals and the analytical determination of the concentration of various metabolites and food components by analytical enzyme techniques.
Enzymes are highly specific in their biological activity and generally catalyze a particular reaction at a very high rate compared to the corresponding reaction occurring at room temperature without biological catalysis. One enzyme may show catalytic activity with respect to a number of substrates upon which it can act. Accordingly, a given enzyme may catalyze the synthesis or degradation of more than one substrate. Some proteins which are not considered classical enzymes, such as bovine serum albumin, show very limited catalytic activity with respect to one or more substrates.
Many enzymes are found in nature in very small quantities. Accordingly, their isolation, purification and use is limited to a small scale operation in view of the expense and time needed to isolate them in a useful form.
Some enzymes occur in nature in relatively large quantities and are relatively easy to isolate, purify and use. Unfortunately, due to the precise catalytic behavior of enzymes, the enzymes available in large quantities can only catalyze certain select reactions. Moreover, many nonenzymatic proteins are available in large supply at low cost in pure form. In the past, these proteins have been useless as catalysts.
Much effort has been directed in the recent past toward the preparation of partially or totally synthetic catalysts which exhibit catalytic behavior similar to the catalytic behavior exhibited by biocatalysts, namely the native enzymes. Many biocatalysts or enzymes are either scarce or expensive to isolate, thus the attempts to mimic their catalytic activity. Further, some attempts have been made to modify native enzymes to change their enzymatic specificity so that they may function to catalyze a reaction which they previously could not catalyze.
2. Description of the References
One technique known to achieve enzyme-like behavior to catalyze a specific reaction is the synthesis of so called enzyme model molecules. For example, low molecular weight compounds may be covalently bonded to functional groups which exhibit the activity of the active site of an enzyme. Examples of such preparations are described in the publications: Breslow, R., Advances in Chemistry Series, R. F. Gould, Ed., American Chemical Society, Washington, D. C. 21-43 (1971) and Tang, C. C.; Davilian, D.; Haung, P. and Breslow, R., J. Amer. Chem. Soc., 100, 3918 (1978) and Breslow, R., Doherty, J., Guillot, G. and Lipsey, C., J. Amer. Chem. Soc., 100, 3227 (1978).
Another technique involves the use of a synthetic polymer matrix which is modified along its backbone to provide functional groups which exhibit the function of the active site of a given enzyme. Examples of such techniques can be found in the following articles: Wulff, G. and Schulza, I., Israel J. Chem., 17, 291 (1978) and Suh, J. and Klotz, I. M., Bioorganic, 6, 165 (1977).
Another technique involves the attachment of a new chemical moiety to a native enzyme near the active site of the enzyme to attempt to cause the enzyme to react with a different catalytic activity. One example of this is the conversion of papain, a proteolytic enzyme to an oxidase type enzyme by the covalent attachment of a flavin near the active site of the native papain enzyme, as illustrated in the articles: Levine, H. L. and Kaiser, E. T., J. Amer. Chem. Soc., 100, 7670 (1978); Kaiser, E. T., et al., Adv. in Chemistry Series, No. 191, Biomimetic Chemistry, 1980; and Otsuki, T.; Nakagawa, Y. and Kaiser, E. T., J. C. S. Chem. Comm., 11,457 (1978). Other examples of such enzymatic modification may be found in the article: Wilson, M. E. and Whitesides, G. M., J. Amer. Chem. Soc., 100, 306 (1978).
Still another attempt to change enzyme specificity is the immobilization of a native enzyme into a gel matrix. For example, trypsin enzyme has been immobilized in polyacrylamide gel. The polyacrylamide gel allows amino acid esters to diffuse through the gel matrix to react with the enzyme but will not allow larger proteins to diffuse through. Thus, the enzyme specificity is changed by eliminating access of one of the substrate molecules to the enzyme.
Also, it is known that a native lysine mono-oxygenase can be reacted to block the sulfhydryl groups on the enzyme. When the specific enzyme lysine mono-oxygenase is so treated, it shows modified catalytic activity toward amino acids and catalyses oxidative deamination instead of its natural oxygenative decarboxylation. However, the reporters cannot account for the modified behavior. See the article by Yamauchi, T.; Yamamoto, S. and Hayaishi, O., in The Journal of Biological Chemistry, 248, 10, 3750-3752 (1973).
Also, it has been reported that by reacting a native enzyme, for example trypsin, with its known inhibitor, and subsequently cross-linking the enzyme, its activity with respect to its typical substrates can be modified. See the article by Beaven, G. H. and Gratzer, W. B. in Int. J. Peptide Res., 5, 215-18 (1973).
Also, synthetic proteins have been synthesized by the anchoring of an amino acid residue on a solid support and subsequently adding amino acid residues one after another.
Further, semisynthetic proteins have been synthesized by a method wherein a native protein is subjected to limited hydroysis to produce protein fragments. The fragments of the native protein are then subjected to a process whereby one or more amino acid residues are added or removed from the fragments to form modified fragments. The resultant modified fragments are then reattached to form the semisynthetic protein with an altered amino acid residue composition. Examples of the synthetic and semisynthetic protein technologies cited immediately above are found in the book Semisynthetic Proteins by R. E. Offord, published by John Wiley and Sons, Ltd., copyrighted in 1980.
While these techniques are suitable for many applications, a need exists for a simple, efficient, economical and systematic method for chemically modifying an inexpensive and commercially availble native protein to produce an enzyme-like modified protein. The protein can show a catalytic enzymatic activity with respect to a desired chemical reaction which was not previously a commercially-useful reaction catalyzed by the native enzyme and which new reaction can be predetermined in a systematic fashion. The methods disclosed in the above-disclosed references simply subject an enzyme to a set of conditions and attempt to eludicate its behavior.